Stabilized immune modulatory rna (simra) compounds

ABSTRACT

The invention relates to the therapeutic use of novel stabilized oligoribonucleotides as immune modulatory agents for immune therapy applications. Specifically, the invention provides novel RNA-based oligoribonucleotides with improved nuclease and RNase stability and that have immune modulatory activity through TLR7 and/or TLR8.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/948,529, filed on Jul. 9, 2007; U.S. Provisional Application Ser.No. 60/957,195, filed on Aug. 22, 2007; U.S. Provisional ApplicationSer. No. 60/981,161, filed on Oct. 19, 2007; and U.S. ProvisionalApplication Ser. No. 61/015,284, filed on Dec. 20, 2007. The contents ofthese applications are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to the field of immunology andimmunotherapy applications using oligoribonucleotides as immunemodulatory agents. More particularly, the invention relates to immunemodulatory RNA compositions and methods of use thereof for modulatingthe immune response through Toll-like receptor 8 (TLR8), Toll-likereceptor 7 (TLR7) and TLR7 and TLR8.

2. Summary of the Related Art

The immune response involves both an innate and an adaptive responsebased upon the subset of cells involved in the response. For example,the T helper (Th) cells involved in classical cell-mediated functionssuch as delayed-type hypersensitivity and activation of cytotoxic Tlymphocytes (CTLs) are Th1 cells, whereas the Th cells involved ashelper cells for B-cell activation are Th2 cells. The type of immuneresponse is influenced by the cytokines and chemokines produced inresponse to antigen exposure. Cytokines provide a means for controllingthe immune response by affecting the balance of T helper 1 (Th1) and Thelper 2 (Th2) cells, which directly affects the type of immune responsethat occurs. If the balance is toward higher numbers of Th1 cells, thena cell-mediated immune response occurs, which includes activation ofcytotoxic T cells (CTLs). When the balance is toward higher numbers ofTh2 cells, then a humoral or antibody immune response occurs. Each ofthese immune response results in a different set of cytokines beingsecreted from Th1 and Th2 cells. Differences in the cytokines secretedby Th1 and Th2 cells may be the result of the different biologicalfunctions of these two T cell subsets.

Th1 cells are involved in the body's innate response to antigens (e.g.viral infections, intracellular pathogens, and tumor cells). The initialresponse to an antigen can be the secretion of IL-12 from antigenpresenting cells (e.g. activated macrophages and dendritic cells) andthe concomitant activation of Th1 cells. The result of activating Th1cells is a secretion of certain cytokines (e.g. IL-2, IFN-gamma andother cytokines) and a concomitant activation of antigen-specific CTLs.Th2 cells are known to be activated in response to bacteria, parasites,antigens, and allergens and may mediate the body's adaptive immuneresponse (e.g. immunoglobulin production and eosinophil activation)through the secretion of certain cytokines (e.g. IL-3, IL-4, IL-5, IL-6,IL-9, IL-10, IL-13 and other cytokines) and chemokines. Secretion ofcertain of these cytokines may result in B-cell proliferation and anincrease in antibody production. In addition, certain of these cytokinesmay stimulate or inhibit the release of other cytokines (e.g IL-10inhibits IFN-γ secretion from Th1 cells and IL-12 from dendritic cells).Ultimately, the balance between Th1 and Th2 cells and the cytokines andchemokines released in response to selected stimulus can have animportant role in how the body's immune system responds to disease. Forexample, IFN-α may inhibit hepatitis C, and MIP-1α and MIP-1 (also knownas CCL3 and CCL4 respectively) may inhibit HIV-1 infection. Optimalbalancing of the Th1/Th2 immune response presents the opportunity to usethe immune system to treat and prevent a variety of diseases.

The Th1 immune response can be induced in mammals for example byintroduction of bacterial or synthetic DNA containing unmethylated CpGdinucleotides, which immune response results from presentation ofspecific oligonucleotide sequences (e.g. unmethylated CpG) to receptorson certain immune cells known as pattern recognition receptors (PRRs).Certain of these PRRs are Toll-like receptors (TLRs).

TLRs are intimately involved in inducing the innate immune response inresponse to microbial infection. In vertebrates, TLRs consist of afamily of ten proteins (TLR1 to TLR10) that are known to recognizepathogen associated molecular patterns. Of the ten, TLR3, 7, 8, and 9are known to localize in endosomes inside the cell and recognize nucleicacids (DNA and RNA) and small molecules such as nucleosides and nucleicacid metabolites. TLR3 and TLR9 are known to recognize nucleic acid suchas dsRNA and unmethylated CpG dinucleotide present in viral andbacterial and synthetic DNA, respectively. Bacterial DNA has been shownto activate the immune system and to generate antitumor activity(Tokunaga T et al., J. Natl. Cancer Inst. (1984) 72:955-962; Shimada S,et al., Jpn. H cancer Res, 1986, 77, 808-816; Yamamoto S, et al., Jpn.J. Cancer Res., 1986, 79, 866-73; Messina, J, et al., J. Immunolo.(1991) 147:1759-1764). Other studies using antisense oligonucleotidescontaining CpG dinucleotides have shown stimulation of an immuneresponse (Zhao Q, et al., Biochem. Pharmacol. 1996, 26, 173-82).Subsequent studies showed that TLR9 recognizes unmethylated CpG motifspresent in bacterial and synthetic DNA (Hemmi H, et al., Nature. (2000)408:740-5). Other modifications of CpG-containing phosphorothioateoligonucleotides can also affect their ability to act through TLR9 andmodulate the immune response (see, e.g., Zhao et al., Biochem.Pharmacol. (1996) 51:173-182; Zhao et al., Biochem Pharmacol. (1996)52:1537-1544; Zhao et al., Antisense Nucleic Acid Drug Dev. (1997)7:495-502; Zhao et al., Bioorg. Med. Chem. Lett. (1999) 9:3453-3458;Zhao et al., Bioorg. Med. Chem. Lett. (2000) 10:1051-1054; Yu et al.,Bioorg. Med. Chem. Lett. (2000) 10:2585-2588; Yu et al., Bioorg. Med.Chem. Lett. (2001) 11:2263-2267; and Kandimalla et al., Bioorg. Med.Chem. (2001) 9:807-813). In addition, structure activity relationshipstudies have allowed identification of synthetic motifs and novelDNA-based structures that induce specific immune response profiles thatare distinct from those resulting from unmethylated CpG dinucleotides.(Kandimalla E R, et al., Proc Natl Acad Sci USA. (2005) 102:6925-30.Kandimalla E R, et al., Proc Natl Acad Sci USA. (2003) 100:14303-8. CongY P, et al., Biochem Biophys Res Commun. (2003) 310:1133-9. Kandimalla ER, et al., Biochem Biophys Res Commun. (2003) 306:948-53. Kandimalla ER, et al., Nucleic Acids Res. (2003) 31:2393-400. Yu D, et al., BioorgMed Chem. (2003) 11:459-64. Bhagat L, et al., Biochem Biophys ResCommun. (2003) 300:853-61. Yu D, et al., Nucleic Acids Res. (2002)30:4460-9. Yu D, et al., J Med Chem. (2002) 45:4540-8. Yu D, et al.,Biochem Biophys Res Commun. (2002) 297:83-90. Kandimalla E R, et al.,Bioconjug Chem. (2002) 13:966-74. Yu D, K et al., Nucleic Acids Res.(2002) 30:1613-9. Yu D, et al., Bioorg Med. Chem. (2001) 9:2803-8. Yu D,et al., Bioorg Med Chem Lett. (2001) 11:2263-7. Kandimalla E R, et al.,Bioorg Med Chem. (2001) 9:807-13. Yu D, et al., Bioorg Med Chem Lett.(2000) 10:2585-8, Putta M R, et al., Nucleic Acids Res. (2006)34:3231-8). However, until recently, natural ligands for TLR7 and TLR8were unknown.

It has been shown that TLRs 7 and 8 recognize viral and syntheticsingle-stranded RNAs and small molecules, including a number ofnucleosides (Diebold, S. S., et al., Science v: 303, 1529-1531 (2004).Diebold et al. (Science, 303:1529-1531 (2004)) show that the IFN-αresponse to influenza virus requires endosomal recognition of influenzagenomic RNA and signaling by means of TLR7 and MyD88 and identify ssRNAas a ligand for TLR7. Certain synthetic compounds, theimidazoquinolones, imiquimod (R-837) and resiquimod (R-848) are ligandsof TLR7 and TLR8 (Hemmi H et al., (2002) Nat Immunol 3:196-200; Jurk Met al., (2002) Nat Immunol 3:499). In addition, certain guanosineanalogs, such as 7-deaza-G, 7-thia-8-oxo-G (TOG), and 7-allyl-8-oxo-G(loxoribine), have been shown to activate TLR7 at high concentrations(Lee J et al., Proc Natl Acad Sci USA. 2003, 100:6646-51). However,these small molecules, eg. imiquimod, are also known to act throughother receptors (Schon M P, et al., (2006) J. Invest Dermatol., 126,1338-47)

The lack of any known specific ssRNA motif for TLR7 or TLR8 recognitionand the potentially wide range of stimulatory ssRNA molecules suggestthat TLR7 and TLR8 can recognize both self and viral RNA. Recently itwas shown that certain GU-rich oligoribonucleotides areimmunostimulatory and act through TLR7 and TLR8 (Heil et al. Science,303: 1526-1529 (2004); Lipford et al. WO03/086280; Wagner et al.WO98/32462) when complexed with N-[1-(2,3-Dioleoyloxy)propyl]-N,N,Ntrimethylammoniummethylsulfate (DOTAP) or other lipid agents. However,RNA molecules have been used for many years, for example as ribozymesand, more recently, siRNA and microRNA, and RNA employed as ribozymes,siRNA, and microRNA contain GU dinucleotides. In addition, a numberthese RNA molecules have been shown to elicit immune responses throughTLR stimulation in the presence of lipids (Kariko et al., Immunity(2005) 23:165-75; Ma Z et al., Biochem Biophys Res Commun., (2005) 330,755-9). However, the instability of these RNA molecules has hinderedprogress in using and applying these molecules in many areas (e.g.prevention and treatment of human disease).

Oligonucleotides and oligodeoxynucleotides containing a ribose ordeoxyribose sugar have been used in a wide variety of fields, includingbut not limited to diagnostic probing, PCR priming, antisense inhibitionof gene expression, siRNA, microRNA, aptamers, ribozymes, andimmunotherapeutic agents based on Toll-like Receptors (TLRs). Morerecently, many publications have demonstrated the use ofoligodeoxynucleotides as immune modulatory agents and their use alone oras adjuvants in immunotherapy applications for many diseases, such asallergy, asthma, autoimmunity, cancer and infectious disease.

The fact that DNA oligonucleotides are recognized by TLR9, while RNAoligonucleotides are recognized by TLR7 and/or TLR8 is most likely dueto differences in the structural conformations between DNA and RNA.However, the chemical differences between DNA and RNA also make DNA farmore chemically and enzymatically stable than RNA.

RNA is rapidly degraded by ubiquitous extracellular ribonucleases(RNases) which ensure that little, if any, self-ssRNA reaches theantigen-presenting cells. Exonuclease degradation of nucleic acids ispredominantly of 3′-nuclease digestion with a smaller percentage through5′-exonuclease action. In addition to exonuclease digestion, RNA canalso be degraded by endonuclease activity of RNAses. RNA-based moleculeshave so far had to be complexed with lipids to provide stability againstnucleases.

While providing an essential function of preventing autoimmunereactivity, these ribonucleases also present a substantial problem forany synthetic ssRNA molecule designed to be exploited for immunotherapy,as ribonucleases will rapidly degrade both synthetic and natural ssRNA.To overcome this hurdle, protection of ssRNA molecules from degradationhas been attempted by encapsulating the ssRNA in lipsomes, condensing itwith polyethylenimine, or complexing it to molecules such as N-[1-(2,3dioleoyloxy)-propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP).However, these protective measures are secondary measures applied to astill unstable ssRNA, and the effects of these protective measures onthe in vivo efficacy and immune modulatory activity of ssRNA (natural orsynthetic) remain unclear.

Agrawal et al. (11/697,422) describe a novel class of SIMRAcompositions. However, a challenge remains to develop additionalcompounds that retain the naked RNA such that it continues to berecognized as a ligand for TLR7 and/or TLR8, while improving itsstability such that it can be made to be a useful in vivo molecule.Ideally, this challenge might be met through the design of inherentlystable RNA-based molecules that can act as new immunotherapic agents,which will find use in a number of clinically relevant applications,such as improving the effects of vaccination when co-administered ortreating and/or preventing diseases when invoking or enhancing an immuneresponse is beneficial, for example cancer, autoimmune disorders, airwayinflammation, inflammatory disorders, infectious diseases, skindisorders, allergy, asthma or diseases caused by pathogens.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the invention provides novel stabilized immunemodulatory RNA (“SIMRA”) compounds, further defined below, and their usefor inducing and/or enhancing an immune response. The novel chemicalentities according to the invention provide immune response inducingand/or enhancing compounds that are substantially more effective atinducing an immune response and substantially less susceptible todegradation. The methods according to the invention enable using SIMRAto modify the cytokine and/or chemokine profile for immunotherapyapplications.

In one embodiment of the first aspect, the invention provides a SIMRAcompound as an agonist for TLR8.

In another embodiment of the first aspect, the invention provides aSIMRA compound as an agonist for TLR7 and TLR8.

In a further embodiment of the first aspect, the invention provides aSIMRA compound as an agonist for TLR7.

In a further embodiment of the first aspect, the invention provides aSIMRA compound as an adjuvant.

In a second aspect, the invention provides pharmaceutical compositions.These compositions comprise any one of the SIMRA compositions of theinvention and a physiologically acceptable or pharmaceuticallyacceptable carrier.

In a third aspect, the invention provides a method for generating animmune response in a vertebrate, the method comprising administering tothe vertebrate at least one of the SIMRA compounds according to theinvention in a pharmaceutically effective amount.

In a fourth aspect, the invention provides a method for therapeuticallytreating a vertebrate having a disease or disorder where inducing and/orenhancing an immune response would be beneficial, for example cancer,autoimmune disorders, airway inflammation, inflammatory disorders,infectious diseases, skin disorders, allergy, asthma or diseases causedby pathogens, such method comprising administering to the patient havingsuch a disorder or disease at least one of the SIMRA compounds accordingto the invention in a pharmaceutically effective amount.

In a fifth aspect, the invention provides a method for preventing adisease or disorder in a vertebrate where inducing and/or enhancing animmune response would be beneficial, for example cancer, an autoimmunedisorder, airway inflammation, inflammatory disorders, infectiousdisease, skin disorders, allergy, asthma or diseases caused by apathogen, such method comprising administering to a vertebrate that issusceptible to such a disorder or disease at least one of the SIMRAcompounds according to the invention in a pharmaceutically effectiveamount.

In a sixth aspect, the invention provides a method of isolating cellscapable of producing cytokine or chemokines (e.g. immune cells, PBMCs),culturing such cells under standard cell culture conditions, ex vivotreating such cells with at least one of the SIMRA compounds of theinvention such that the isolated cells produce or secrete increasedlevels of cytokines or chemokines, and administering or re-administeringthe treated cells to a patient in need of cytokine or chemokine therapyfor the prevention or treatment of disease.

In a further embodiment of this aspect of the invention, the patient inneed of cytokine or chemokine therapy for prevention or treatment ofdisease is administered the isolated, SIMRA-treated cells in combinationwith one or more SIMRA compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

SIMRA compounds of the invention were synthesized according toExample 1. FIG. 1 is a synthetic scheme for the parallel synthesis ofSIMRA compounds of the invention. DMTr=4,4′-dimethoxytrityl;CE=cyanoethyl.

FIG. 2A depicts NF-κB activity in HEK293 cells expressing human TLR8that were treated and analyzed according to example 2. Briefly, theHEK293 cells were stimulated with 150 μg/ml of agonists of TLR8 for 18hr, and the levels of NF-κB were determined using SEAP (secreted form ofhuman embryonic alkaline phosphatase) assay. The data demonstrate thatadministration of a SIMRA according to the invention generates adistinct TLR-mediated immune response profile.

FIG. 2B depicts NF-κB activity in HEK293 cells expressing human TLR8that were treated and analyzed according to example 2. Briefly, theHEK293 cells were stimulated with 150 μg/ml of agonists of TLR8 for 20hr and the levels of NF-κB were determined using SEAP assay. The datademonstrate that administration of a SIMRA according to the inventiongenerates a distinct TLR-mediated immune response profile.

FIG. 2C-2E depict NF-κB activity in HEK293 cells expressing human TLR8that were treated and analyzed according to example 2. Briefly, theHEK293 cells were stimulated with 150 μg/ml of agonists of TLR8 for 18hr, and the levels of NF-κB were determined using SEAP assay. The datademonstrate that administration of a SIMRA according to the inventiongenerates a distinct TLR-mediated immune response profile.

FIG. 2F depicts NF-κB activity in HEK293 cells expressing human TLR8that were treated and analyzed according to example 2. Briefly, HEK293cells expressing human TLR8 were stimulated with 0, 20, 50, 100, 200, or300 μg/ml of agonists for 18 hr. The levels of NF-κB were determinedusing SEAP assay. The data demonstrate that administration of a SIMRAaccording to the invention generates a distinct TLR-mediated immuneresponse profile.

FIG. 2G depicts NF-κB activity in HEK293 cells expressing human TLR7that were treated and analyzed according to example 2. Briefly, HEK293cells expressing human TLR7 were stimulated with 0, 20, 50, 100, 200, or300 μg/ml of agonists for 18 hr. The levels of NF-κB were determinedusing SEAP assay. The data demonstrate that administration of a SIMRAaccording to the invention generates a distinct TLR-mediated immuneresponse profile.

FIGS. 2H, 2J and 2L depict NF-κB activity in HEK293 cells expressinghuman TLR8 that were treated and analyzed according to example 2.Briefly, HEK293 cells expressing human TLR8 were stimulated with 0, 20,50, 100, 200, or 300 μg/ml of agonists for 18 hr. The levels of NF-κBwere determined using SEAP assay. The data demonstrate thatadministration of a SIMRA according to the invention generates adistinct TLR-mediated immune response profile.

FIGS. 2I, 2K and 2M depict NF-κB activity in HEK293 cells expressinghuman TLR7 that were treated and analyzed according to example 2.Briefly, HEK293 cells expressing human TLR7 were stimulated with 0, 20,50, 100, 200, or 300 μg/ml of agonists for 18 hr. The levels of NF-κBwere determined using SEAP assay. The data demonstrate thatadministration of a SIMRA according to the invention generates adistinct TLR-mediated immune response.

FIGS. 3A-3C depict cytokine and chemokine concentrations from humanPBMCs that were treated and analyzed according to example 3. Briefly,the PBMCs were isolated from freshly obtained healthy human volunteer'sblood and cultured with 50 μg/ml dose of TLR7/8 agonists for 24 hr, andsupernatants were collected and analyzed by Luminex multiplex assaycytokine and chemokine levels. The data demonstrate that administrationof a SIMRA according to the invention generates a distinct TLR-mediatedcytokine and chemokine profile.

FIGS. 4A-4C depict cytokine and chemokine concentrations from humanPBMCs that were treated and analyzed according to example 3. Briefly,the PBMCs were isolated from freshly obtained healthy human volunteer'sblood and cultured with 200 μg/ml dose of TLR7/8 agonists for 24 hr, andsupernatants were collected and analyzed by Luminex multiplex assaycytokine and chemokine levels. The data demonstrate that administrationof a SIMRA according to the invention generates a distinct TLR-mediatedcytokine and chemokine profile.

FIGS. 4D-4AA depict cytokine and chemokine concentrations from humanPBMCs that were treated and analyzed according to example 3. Briefly,the PBMCs were isolated from freshly obtained healthy human volunteer'sblood and cultured with increasing concentrations of TLR7/8 agonists for24 hr, and supernatants were collected and analyzed by Luminex multiplexassay cytokine and chemokine levels. The data demonstrate thatadministration of a SIMRA according to the invention generates adistinct, dose-dependent, TLR-mediated cytokine and chemokine profile.

FIGS. 5A-5C depict cytokine and chemokine concentrations from humanplasmacytoid dendritic cells (pDCs) that were isolated, treated, andanalyzed according to example 3. Briefly, the pDCs were isolated fromfreshly obtained healthy human volunteer's blood PBMCs and cultured with50 μg/ml dose of TLR7/8 agonists for 24 hr, and supernatants werecollected and analyzed by Luminex multiplex assay for cytokine andchemokine levels. The data demonstrate that administration of a SIMRAaccording to the invention generates a distinct TLR-mediated cytokineand chemokine profile.

FIG. 5D depicts cytokine and chemokine concentrations from humanplasmacytoid dendritic cells (pDCs) that were isolated, treated, andanalyzed according to example 3. Briefly, the pDCs were isolated fromfreshly obtained healthy human volunteer's blood PBMCs and cultured witha 50 μg/ml or 200 μg/ml dose of TLR7/8 agonists for 24 hr, andsupernatants were collected and analyzed by Luminex multiplex assay forcytokine and chemokine levels. The data demonstrate that administrationof a SIMRA according to the invention generates a distinct TLR-mediatedcytokine and chemokine profile.

FIGS. 6A, 6B, 6C, 6E and 6F depict cytokine and chemokine concentrationsfrom human plasmacytoid dendritic cells (pDCs) that were treated andanalyzed according to example 3. Briefly, the pDC were isolated fromfreshly obtained healthy human volunteer's blood PBMCs and cultured with200 μg/ml dose of TLR7/8 agonists for 24 hr, and supernatants werecollected and analyzed by Luminex multiplex assay for cytokine andchemokine levels. The data demonstrate that administration of a SIMRAaccording to the invention generates a distinct TLR-mediated cytokineand chemokine profile.

FIG. 6D depicts cytokine and chemokine concentrations from humanplasmacytoid dendritic cells (pDCs) that were treated and analyzedaccording to example 3. Briefly, the pDC were isolated from freshlyobtained healthy human volunteer's blood PBMCs and cultured with 100μg/ml dose of TLR7/8 agonists for 24 hr, and supernatants were collectedand analyzed by Luminex multiplex assay for cytokine and chemokinelevels. The data demonstrate that administration of a SIMRA according tothe invention generates a distinct TLR-mediated cytokine and chemokineprofile.

FIGS. 7A-7C depict cytokine and chemokine concentrations from humanmyeloid dendritic cells (mDCs) that were treated and analyzed accordingto example 3. Briefly, the mDCs were isolated from freshly obtainedhealthy human volunteer's blood PBMCs and cultured with 50 μg/ml dose ofTLR7/8 agonists for 24 hr, and supernatants were collected and analyzedby Luminex multiplex assay for cytokine and chemokine levels. The datademonstrate that administration of a SIMRA according to the inventiongenerates a distinct TLR-mediated cytokine and chemokine profile.

FIGS. 8A and 8B depict serum cytokine induction in C57BL/6 mice (n=3) 2hours after they were treated and analyzed according to example 4.Briefly, the C57BL/6 mice were injected subcutaneously with 25 mg/kgdose of TLR7/8 agonists, and 2 hours after administration of theagonist, serum was analyzed for cytokine and chemokine levels, and IL-12levels are presented. The data demonstrate that in vivo administrationof a SIMRA according to the invention generates a distinct TLR-mediatedcytokine and chemokine profile.

FIGS. 9A and 9B depict serum cytokine induction in BALB/c mice (n=3) 2hours after they were treated and analyzed according to example 4.Briefly, the BALB/c mice injected subcutaneously with 25 mg/kg dose ofTLR7/8 agonists, and 2 hours after administration of the agonist, serumwas analyzed for cytokine and chemokine levels, and IL-12 levels arepresented. The data demonstrate that in vivo administration of a SIMRAaccording to the invention generates a distinct TLR-mediated cytokineand chemokine profile.

FIGS. 9C-9F depict serum cytokine induction in BALB/c mice (n=3) 2 hoursafter they were treated and analyzed according to example 4. Briefly,the BALB/c mice injected subcutaneously with 10 mg/kg or 25 mg/kg doseof TLR7/8 agonists, and 2 hours after administration of the agonist,serum was analyzed for cytokine and chemokine levels, and IL-12 levelsare presented. The data demonstrate that in vivo administration of aSIMRA according to the invention generates a distinct TLR-mediatedcytokine and chemokine profile.

FIGS. 10A-10H depict serum stability of exemplar SIMRA compounds fromTable 2 that were treated according to example 5. Briefly, approximately0.5 OD of exemplar SIMRA compounds were individually incubated in 1%human serum in PBS for 30 minute at 37° C. At the end of the 30 minuteincubation, the SIMRA compound was analyzed on anion-exchange HPLC todetermine the percentage of full-length SIMRA compound that remained ascompared to the amount of SIMRA compound present before serum treatment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to the therapeutic use of oligoribonucleotides asimmune modulatory agents for immunotherapy applications. Specifically,the invention provides RNA-based oligonucleotides with improved in vivostability that modulate the immune response through TLR7 alone, TLR7 andTLR8 or TLR8 alone (SIMRA compounds). By initiating diverse innate andacquired immune response mechanisms, for example through activation ofdendritic cells and other antigen-presenting cells with stable agonistsof TLR7 and/or TLR8, or SIMRA compounds, the resulting cytokine profilecan lead to the destruction of pathogens, infected cells or tumor cellsand development of antigen-specific antibody and CTL responses. Thus,the invention provides a diverse set of SIMRA compounds, each having itsown unique immune regulatory characteristics. In this way, the scope andnature of the immune response can be customized for distinct medicalindications by providing the SIMRA compound having the desired set ofimmune modulatory characteristics for that indication. The issuedpatents, patent applications, and references that are cited herein arehereby incorporated by reference to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.In the event of inconsistencies between any teaching of any referencecited herein and the present specification, the latter shall prevail forpurposes of the invention.

The invention provides methods for using SIMRA compounds to enhance theimmune response. Such methods will find use in immunotherapyapplications such as, but not limited to, treatment of cancer,autoimmune disorders, asthma, respiratory allergies, food allergies,skin allergies, and bacteria, parasitic, and viral infections in adultand pediatric human and veterinary applications. Thus, the inventionfurther provides novel SIMRA compounds having optimal levels of immunemodulatory effect for immunotherapy and methods for making and usingsuch compounds. In addition, SIMRA compounds of the invention are usefulas adjuvants or in combination with an agent useful for treating thedisease or condition that does not diminish the immune modulatory effectof the SIMRA compound for prevention and treatment of diseases.

DEFINITIONS

The term “2′-substituted ribonucleoside” or “2′-substituted arabinoside”generally includes ribonucleosides or arabinonucleosides in which thehydroxyl group at the 2′ position of the pentose moiety is substitutedto produce a 2′-substituted or 2′-O-substituted ribonucleoside. Incertain embodiments, such substitution is with a lower hydrocarbyl groupcontaining 1-6 saturated or unsaturated carbon atoms, with a halogenatom, or with an aryl group having 6-10 carbon atoms, wherein suchhydrocarbyl, or aryl group may be unsubstituted or may be substituted,e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy,alkoxy, carboxyl, carboalkoxy, or amino groups. Arabinonucleosides ofthe invention include, but are not limited to, arabino-G, arabino-C,arabino-U, arabino-A. Examples of 2′-O-substituted ribonucleosides or2′-O-substituted-arabinosides include, without limitation 2′-amino,2′-fluoro, 2′-allyl, 2′-O-alkyl and 2′-propargyl ribonucleosides orarabinosides, 2′-O-methylribonucleosides or 2′-O-methylarabinosides and2′-O-methoxyethoxyribonucleosides or 2′-O-methoxyethoxyarabinosides.

The term “3′”, when used directionally, generally refers to a region orposition in a polynucleotide or oligonucleotide 3′ (toward the 3′position of the sugar) from another region or position in the samepolynucleotide or oligonucleotide.

The term “5′”, when used directionally, generally refers to a region orposition in a polynucleotide or oligonucleotide 5′ (toward the 5′position of the sugar) from another region or position in the samepolynucleotide or oligonucleotide.

The term “about” generally means that the exact number is not critical.Thus, the number of ribonucleoside residues in the oligoribonucleotidesis not critical, and oligoribonucleotides having one or two fewerribonucleoside or arabinonucleoside residues, or from one to severaladditional ribonucleoside or arabinonucleoside residues are contemplatedas equivalents of each of the embodiments described above.

The term “adjuvant” generally refers to a substance which, when added toan immunogenic agent such as vaccine or antigen, enhances or potentiatesan immune response to the agent in the recipient host upon exposure tothe mixture.

The term “airway inflammation” generally includes, without limitation,inflammation in the respiratory tract caused by infectious allergens,including asthma.

The term “allergen” generally refers to an antigen or antigenic portionof a molecule, usually a protein, which elicits an allergic responseupon exposure to a subject. Typically the subject is allergic to theallergen as indicated, for instance, by the wheal and flare test or anymethod known in the art. A molecule is said to be an allergen even ifonly a small subset of subjects exhibit an allergic (e.g., IgE) immuneresponse upon exposure to the molecule.

The term “allergy” generally includes, without limitation, foodallergies, respiratory allergies, and skin allergies.

The term “antigen” generally refers to a substance that is recognizedand selectively bound by an antibody or by a T cell antigen receptor.Antigens may include but are not limited to peptides, proteins,nucleosides, nucleotides, and combinations thereof. Antigens may benatural or synthetic and generally induce an immune response that isspecific for that antigen.

The term “autoimmune disorder” generally refers to disorders in which“self” antigen undergo attack by the immune system.

Blocking 3′ or 5′ degradation or “cap” or “capping” means that the 3′ or5′ end of the oligoribonucleotide is attached to another molecule (e.glinker or other non-RNA nucleotide) to sufficiently inhibit nucleasedegradation (e.g. 3′ exonuclease degradation).

The term “carrier” generally encompasses any excipient, diluent, filler,salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containingvesicle, microspheres, liposomal encapsulation, or other material wellknown in the art for use in pharmaceutical formulations. It will beunderstood that the characteristics of the carrier, excipient, ordiluent will depend on the route of administration for a particularapplication. The preparation of pharmaceutically acceptable formulationscontaining these materials is described in, e.g., Remington'sPharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack PublishingCo., Easton, Pa., 1990.

The term “co-administration” generally refers to the administration ofat least two different substances sufficiently close in time to modulatean immune response. Co-administration includes simultaneousadministration of at least two different substances.

The term “complementary” generally means having the ability to hybridizeto a nucleic acid. Such hybridization is ordinarily the result ofhydrogen bonding between complementary strands, preferably to formWatson-Crick or Hoogsteen base pairs, although other modes of hydrogenbonding, as well as base stacking can also lead to hybridization.

The term “immune modulatory oligoribonucleotide” generally refers to anoligoribonucleotide that induces or represses an immune response whenadministered to a vertebrate, such as a fish, fowl or mammal.

The term “in combination with” generally means in the course of treatingthe same disease in the same patient, and includes administering a SIMRAcompound and an agent useful for treating the disease or condition thatdoes not diminish the immune modulatory effect of the SIMRA compound inany order, including simultaneous administration or co-administration,as well as temporally spaced order from a few seconds up to several daysapart. Such combination treatment may also include more than a singleadministration of a SIMRA compound, and/or independently the agent. Theadministration of the SIMRA compound and the agent may be by the same ordifferent routes.

The term “individual” or “subject” generally refers to a mammal, such asa human. Mammals generally include, but are not limited to, humans,non-human primates, rats, mice, cats, dogs, horses, cattle, cows, pigs,sheep, and rabbits.

The term “linear synthesis” generally refers to a synthesis that startsat one end of the immune modulatory oligoribonucleotide and progresseslinearly to the other end. Linear synthesis permits incorporation ofeither identical or non-identical (in terms of length, base compositionand/or chemical modifications incorporated) monomeric units into theimmune modulatory oligoribonucleotides.

The term “linker” generally refers to any moiety that can be attached toan oligoribonucleotide by way of covalent or non-covalent bondingthrough a sugar, a base, or the backbone. The linker can be used toattach two or more nucleosides or can be attached to the 5′ and/or 3′terminal nucleotide in the oligoribonucleotide. Such linker can beeither a non-nucleotidic linker or a nucleotidic linker.

The term “modified nucleoside” generally is a nucleoside that includes amodified heterocyclic base, a modified sugar moiety, or any combinationthereof. In some embodiments, the modified nucleoside is a non-naturalpyrimidine or purine nucleoside, as herein described. For purposes ofthe invention, a modified nucleoside, a pyrimidine or purine analog ornon-naturally occurring pyrimidine or purine can be used interchangeablyand refers to a nucleoside that includes a non-naturally occurring baseand/or non-naturally occurring sugar moiety. For purposes of theinvention, a base is considered to be non-natural if it is not guanine,cytosine, adenine or uracil. In some embodiments, the modifiednucleoside is a 2′-substituted ribonucleoside an arabinonucleoside or a2′-deoxy-2′-substituted-arabinoside that can be substituted intoselected positions of the oligoribonucleotide to improve stabilitywithout interfering with TLR7 or TLR8 activity.

The term “modulation” or “stimulation” generally refers to change, suchas an increase in a response or qualitative difference in a response,which can arise from eliciting and/or enhancement of a response.

The term “non-nucleotidic linker” generally refers to a chemical moietyother than a nucleotidic linkage that can be attached to anoligoribonucleotide by way of covalent or non-covalent bonding.Preferably such non-nucleotidic linker is from about 2 angstroms toabout 200 angstroms in length, and may be either in a cis or transorientation.

The term “nucleotidic linkage” generally refers to a chemical linkage tojoin two nucleosides through their sugars (e.g. 3′-3′,2′-3′,2′-5′,3′-5′)consisting of a phosphate, non-phosphate, charged, or neutral group(e.g., phosphodiester, phosphorothioate or phosphorodithioate) betweenadjacent nucleosides.

The term “peptide” generally refers to polypeptides that are ofsufficient length and composition to affect a biological response, e.g.,antibody production or cytokine activity whether or not the peptide is ahapten. The term “peptide” may include modified amino acids (whether ornot naturally or non-naturally occurring), where such modificationsinclude, but are not limited to, phosphorylation, glycosylation,pegylation, lipidization, and methylation.

The terms “pharmaceutically acceptable” or “physiologically acceptable”generally refer to a material that does not interfere with theeffectiveness of a compound according to the invention and that iscompatible with a biological system such as a cell, cell culture,tissue, or organism. Preferably, the biological system is a livingorganism, such as a vertebrate.

The term a “pharmaceutically effective amount” generally refers to anamount sufficient to affect a desired biological effect, such as abeneficial result. Thus, a “pharmaceutically effective amount” willdepend upon the context in which it is being administered. Apharmaceutically effective amount may be administered in one or moreprophylactic or therapeutic administrations.

The term “SIMRA” generally refers to stabilized immune modulatory RNAcompounds which are recognized as ligands by TLR7 and/or TLR8, whereinthe compounds may contain single-stranded RNA (ssRNA) and/ordouble-stranded RNA (dsRNA), and modifications to protect or stabilizeits 3′ ends (e.g. by blocking 3′ degradation or by capping the 3′ endsor by linking the 3′ ends of two or more oligoribonucleotides), providedthat the SIMRA is or would be more stable in vivo than an unmodifiedoligoribonucleotide and, thus, affect its immune modulatorycapabilities. The SIMRA may contain modified oligoribonucleotides. TheSIMRA compound may also contain modifications to protect its 5′ ends(e.g., by blocking 5′ degradation or capping the 5′ ends) to furtherimprove the stability of the oligoribonucleotide(s). The SIMRA can belinear or branched, with nucleic acids being polymers of ribonucleosideslinked through, for example, phosphodiester, phosphorothioate, oralternate linkages. A SIMRA may consist of a purine (adenine (A) orguanine (G) or derivatives thereof (e.g. 7-deaza-G, arabino-G andarabino-A)) or pyrimidine (cytosine (C) or uracil (U), or derivativesthereof (e.g. arabino-C and arabino-U)) base covalently attached to aribose sugar residue or a derivative thereof.

The term “treatment” generally refers to an approach intended to obtaina beneficial or desired result, which may include alleviation ofsymptoms, or delaying or ameliorating a disease progression.

The term “viral disease” generally refers to a disease that has a virusas its etiologic agent, including but not limited to hepatitis B,hepatitis C, influenza, acquired immunodeficiency syndrome (AIDS), andherpes zoster.

In a first aspect, the invention provides novel SIMRA compounds. Thepresent inventors have discovered that modification of an immunemodulatory oligoribonucleotide to protect its 3′ end (e.g. by blocking3′ degradation or capping the 3′ end or by linking the 3′ ends of two ormore oligoribonucleotides) surprisingly affects its immune modulatorycapabilities. In addition, it has been determined that this protectionsurprisingly improves the stability of the oligoribonucleotides,removing the need for lipid association or other means of protection.Further, blocking 5′ degradation or capping the 5′ end in addition to orin combination with protecting the 3′-end can also improve the stabilityof the oligoribonucleotide.

In the present invention activation of TLR8 and induction of uniqueimmune responses (e.g. changes in cytokine and/or chemokine profiles)with novel SIMRA compounds is demonstrated. Moreover, the incorporationof certain chemical modification(s) in such human TLR8 activating RNAscan also activate TLR7, resulting in distinct immune response(s) and achange in cytokine and/or chemokine profiles. Thus, the presentinventors have surprisingly discovered that through activation of TLR8and/or TLR7 cytokine and/or chemokine profiles associated therewith canbe modulated by using modified chemical structures, including modifiedbases, modified sugars, backbone, linkers, linkages, and/or caps as partof an immune modulatory oligoribonucleotide.

In one embodiment, the invention provides an immune modulatory compoundcomprising at least two RNA-based oligonucleotides linked at their 3′ends, or an internucleoside linkage or a functionalized nucleobase orsugar to a non-nucleotidic linker. Such embodiment of the invention mayhave at least one accessible 5′ end, which may be capped or uncapped. Ithas been determined that this structure provides further stability (e.g.inhibition of exonuclease activity) to the SIMRA compounds without theneed for lipid association or other protection. An “accessible 5′ end”means that the 5′-terminus of the SIMRA is not modified in such a way asto prevent the SIMRA compound from modulating an immune response throughTLR7 and/or TLR8.

In another embodiment of this aspect of the invention comprises at leasttwo oligoribonucleotides, wherein the immune modulatory compound has astructure including, but not limited to, those as detailed in FormulasI-X in Table 1.

TABLE 1 Oligoribonucleotide Formulas I-X Formula I

Formula II a

Formula II b

Formula III

Formula IV

Formula V

Formula VI

Formula VII

Formula VIII

Formula IX

Formula X

Domains A, B, C, and D may be independently from about 2 to about 35ribonucleotides, and in some embodiments from about 2 to about 20, orfrom about 2 to about 12, or from about 2 to about 11 or from about 2 toabout 8 ribonucleotides in length. Domains A, B, C, and/or D may or maynot be identical. Domains A, B, C, and D may independently be 5′-3′ or2′-5′ RNA having or not having a self-complementary domain, a homo orhetero ribonucleotide sequence, or a linker. “n” may be from 1 to anunlimited number.

“X” is a linker joining or capping Domains A, B, C, and/or D that may bethrough a 3′ or 5′ linkage, a phosphate group, a nucleobase, a non-RNAnucleotide, or a non-nucleotidic linker that may be aliphatic, aromatic,aryl, cyclic, chiral, achiral, a peptide, a carbohydrate, a lipid, afatty acid, mono-tri- or hexapolyethylene glycol, or a heterocyclicmoiety, or combinations thereof.

In a further embodiment, the invention provides a SIMRA compoundcomprising at least two oligoribonucleotides linked by a non-nucleotidiclinker, wherein the sequences of the immune modulatoryoligoribonucleotides may be at least partially self-complementary. Aswould be recognized by one skilled in the art, the complementarysequence of the oligoribonucleotides allows for intermolecular hydrogenbonding thereby giving the oligoribonucleotides secondary structure.Additional oligoribonucleotides can bind together thereby creating achain, or multimers, of oligoribonucleotides according to the invention.

Similar considerations apply to intermolecular base pairing betweenimmune modulatory oligoribonucleotides of different base sequence. Thus,where a plurality of immune modulatory oligoribonucleotides is usedtogether, the plurality of immune modulatory oligoribonucleotides may,but need not, include sequences that are at least partiallycomplementary to one another. In one embodiment the plurality of immunemodulatory oligoribonucleotides includes an immune modulatoryoligoribonucleotide having a first sequence and an immune modulatoryoligoribonucleotide having a second sequence, wherein the first sequenceand the second sequence are at least 50 percent complementary. Forexample, as between two 8-mers that are at least 50 percentcomplementary, they may form 4, 5, 6, 7, or 8 G-C, A-U, and/or G-Uwobble basepairs. Such basepairs may, but need not necessarily, involvebases located at either end of the complementary immune modulatoryoligoribonucleotides. The degree of complementarity may depend on thealignment between immune modulatory oligoribonucleotides, and suchalignment may or may not include single- or multiple-nucleosideoverhangs. In other embodiments, the degree of complementarity is atleast 60 percent, at least 70 percent, at least 80 percent, at least 90percent, or even 100 percent.

As would be recognized by one skilled in the art, the depicted immunemodulatory compounds may have secondary structure because the sequencesof the domains are complementary allowing for intermolecular hydrogenbonding. Moreover, as can be imagined from Formulas I through X,additional linked RNA-based oligonucleotides can bind throughintermolecular hydrogen bonding thereby creating a chain, or multimers,wherein any number of linked RNA-based oligonucleotides may beincorporated.

In another embodiment, the invention provides an immune modulatorycompound comprising at least two RNA-based oligonucleotides linked attheir 3′ or 5′ ends, or through an internucleoside linkage or afunctionalized nucleobase or sugar to a non-nucleotidic linker, andwherein a linker (e.g. cap) is attached to at least one 5′ end. It hasbeen determined that this structure provides further stability (e.g.inhibition of exonuclease activity) to the SIMRA compounds. The5′-terminus of the SIMRA is not modified in such a way as to prevent theSIMRA compound from modulating an immune response through TLR7 and/orTLR8.

In some embodiments, the oligoribonucleotides each independently havefrom about 2 to about 35 ribonucleoside residues. Thus in certainembodiments the oligoribonucleotide can independently be 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 ribonucleotides long.Preferably the oligoribonucleotide is from about 4 to about 30ribonucleoside residues, more preferably from about 4 to about 20ribonucleoside residues or from about 4 to about 11 ribonucleosideresidues. In some embodiments, the immune modulatoryoligoribonucleotides comprise oligoribonucleotides having from about 1to about 18, or from about 1 to about 11, or from about 5 to about 14ribonucleoside residues. In some embodiments, one or more of theoligoribonucleotides have 11 ribonucleotides or from about 8 to about 14ribonucleotides or from about 10 to about 12 ribonucleotides. In thecontext of immune modulatory oligoribonucleotides, preferred embodimentshave from about 1 to about 35 ribonucleotides, preferably from about 5to about 26 ribonucleotides, more preferably from about 13 to about 26ribonucleotides. Preferably, the immune modulatory oligoribonucleotidecomprises at least one phosphodiester, phosphorothioate, orphosphorodithioate interribonucleoside linkage.

In exemplar embodiments each ribonucleoside unit includes a heterocyclicbase and a pentofuranosyl, trehalose, arabinose, 2′-deoxy-2′-substitutedarabinose, 2′-O-substituted ribose or arabinose, or hexose sugar group.The ribonucleoside residues can be coupled to each other by any of thenumerous known interribonucleoside linkages. Such interribonucleosidelinkages include, without limitation, phosphodiester, phosphorothioate,phosphorodithioate, alkylphosphonate, alkylphosphonothioate,phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy,acetamidate, carbamate, morpholino, borano, thioether, bridgedphosphoramidate, bridged methylene phosphonate, bridgedphosphorothioate, and sulfone interribonucleoside linkages. Possiblesites of conjugation for the ribonucleotide are indicated in Formula XI,below, wherein B represents a heterocyclic base.

The SIMRA compounds of the invention can include naturally occurringribonucleosides, modified ribonucleosides, or mixtures thereof.

In the present invention, novel SIMRA compounds are recognized by humanTLR8 and incorporation of certain chemical modification(s) in such humanTLR8 activating RNAs can causes them to be recognized by human TLR7 andinduce immune responses. Such chemical modifications include, but arenot limited to, guanine analogues such as 7-deaza-G, ara-G, 6-thio-G,Inosine, Iso-G, loxoribine, TOG(7-thio-8-oxo)-G, 8-bromo-G, 8-hydroxy-G,5-aminoformycin B, Oxoformycin, 7-methyl-G, 9-p-chlorophenyl-8-aza-G,9-phenyl-G, 9-hexyl-guanine, 7-deaza-9-benzyl-G,6-Chloro-7-deazaguanine, 6-methoxy-7-deazaguanine, 8-Aza-7-deaza-G(PPG),2-(Dimethylamino)guanosine, 7-Methyl-6-thioguanosine,8-Benzyloxyguanosine, 9-Deazaguanosine, and1-(B-D-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine. Chemicalmodifications also include, but are not limited to, adenine analoguessuch as 9-benzyl-8-hydroxy-2-(2-methoxyethoxy)adenine, 2-Amino-N2-O—,methyladenosine, 8-Aza-7-deaza-A, 7-deaza-A, ara-A, Vidarabine,2-Aminoadenosine, N1-Methyladenosine, 8-Azaadenosine, 5-Iodotubercidin.Chemical modifications also include, but are not limited to, cytosineand uracil analogues such as pseudouridine, ara-C, ara-U,5-methylcytidine, 4-thiouridine, N4-ethyluridine, zebularine,5-aminoallyluridine, N3-methyluridine, 5-fluorouridine.

The “immune modulatory oligoribonucleotides” according to the inventionare SIMRA compounds that comprise at least two oligoribonucleotideslinked covalently or non-covalently at their 3′- or 2′-ends orfunctionalized ribose or functionalized ribonucleobase via anon-nucleotidic or a nucleotidic linker. Several examples of linkers areset forth below. Non-covalent linkages include, but are not limited to,electrostatic interaction, hydrophobic interactions, π-stackinginteractions and hydrogen bonding.

In yet other embodiments, the non-nucleotidic linker is an organicmoiety having functional groups that permit attachment to theoligoribonucleotide. Such attachment preferably is by a stable covalentlinkage. As a non-limiting example, the linker may be attached to anysuitable position on the nucleotide. In some preferred embodiments, thelinker is attached to the 3′-hydroxyl. In such embodiments, the linkerpreferably comprises a hydroxyl functional group, which preferably isattached to the 3′-hydroxyl by means of a phosphate-based linkage like,phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonateor non-phosphate-based linkages.

In some embodiments, the non-nucleotidic linker is a small molecule,macromolecule or biomolecule, including, without limitation,polypeptides, antibodies, lipids, antigens, allergens, andoligosaccharides. In some other embodiments, the non-nucleotidic linkeris a small molecule. For purposes of the invention, a small molecule isan organic moiety having a molecular weight of less than 1,000 Da. Insome embodiments, the small molecule has a molecular weight of less than750 Da.

In some embodiments, the small molecule is an aliphatic or aromatichydrocarbon, either of which optionally can include, either in thelinear chain connecting the oligoribonucleotides or appended to it, oneor more functional groups including, but not limited to, hydroxy, amino,thiol, thioether, ether, amide, thioamide, ester, urea, or thiourea. Thesmall molecule can be cyclic or acyclic. Examples of small moleculelinkers include, but are not limited to, amino acids, carbohydrates,cyclodextrins, adamantane, cholesterol, haptens and antibiotics.However, for purposes of describing the non-nucleotidic linker, the term“small molecule” is not intended to include a nucleoside.

In some embodiments, the non-nucleotidic linker is an alkyl linker oramino linker. The alkyl linker may be branched or unbranched, cyclic oracyclic, substituted or unsubstituted, saturated or unsaturated, chiral,achiral or racemic mixture. The alkyl linkers can have from about 2 toabout 18 carbon atoms. In some embodiments such alkyl linkers have fromabout 3 to about 9 carbon atoms. Some alkyl linkers include one or morefunctional groups including, but not limited to, hydroxy, amino, thiol,thioether, ether, amide, thioamide, ester, urea, and thioether. Suchalkyl linkers can include, but are not limited to, 1-propanol, 1,2propanediol, 1,3 propanediol, 1,2,3, propanetriol, triethylene glycol,hexaethylene glycol, polyethylene glycol linkers (e.g. [—O—CH2-CH2-].(n=1-9)), methyl linkers, ethyl linkers, propyl linkers, butyl linkers,or hexyl linkers. In some embodiments, such alkyl linkers may includepeptides or amino acids.

In some embodiments, the non-nucleotidic linker may include, but are notlimited to, those listed in Table 2.

TABLE 2 Representative Non-nucleotidic Linkers

Glycerol (1,2,3-Propanetriol)

1,1,1-Tris(hydroxymethyl)nitromethane

1,2,4-Butanetriol

1,1,1-Tris(hydroxymethyl)propane

2-(hydroxymethyl)-1,3-propanediol

1,2,6-Hexanetriol

2-(hydroxymethyl)1,4-butanediol

3-Methyl-1,3,5-pentanetriol

1,3,5-Pentanetriol

1,2,3-Heptanetriol

1,1,1-Tris(hydroxymethyl)ethane

2-Amino-2-(hydroxymethyl)-1,3-propanediol

N-[Tris(hydroxymethyl)methyl]acrylamide

cis-1,3,5-Cyclohexanetriol

1,3-Di(hydroxyethyoxy)-2-hydroxy-propane

cis-1,3,5-Tri(hydroxymethyl)cyclohexane

1,3-Di(hydroxypropoxy)-2-hydroxyl-propane

1,3,5,-Trihydroxyl-benzene

2-Deoxy-D-ribose

3,5,-Di(hydroxymethyl)phenol

1,2,4,-Trihydroxyl-benzene

1,3,5,-Tri(hydroxymethyl)benzene

D-Galactoal

1,6-anhydro-β-D-Glucose

4,6-Nitropyrogallol

1,3,5-Tris(2-hydroxyethyl)-Cyanuric acid

Gallic acid

3,5,7-Trihydroxyflavone

Ethylene glycol

1,5-Pentanediol

1,3-Propanediol

2,4-Pentanediol

1,2-Propanediol

1,6-Hexanediol

1,4-Butanediol

1,2-Hexanediol

1,3-Butanediol

1,5-Hexanediol

2,3-Butanediol

2,5-Hexanediol

1,4-Butanediol

1,7-Heptanediol

2-(1-Aminopropyl)-1,3-propanediol

1,8-Octanediol

1,2-Dideoxyribose

1,2-Octanediol

1,9-Nonanediol

1,12-Dodecanediol

Triethylene glycol

Tetraethylene glycol

Hexaethylene glycol

Cis, cis-cyclohexanetriol linker

Cis, trans-cyclohexanetriol linker

1,3,4-Isobutanetriol

Cyanuric acid

In some embodiments, the small molecule linker is glycerol or a glycerolhomolog of the formula HO—(CH₂)_(o)—CH(OH)—(CH₂)_(p)—OH, wherein o and pindependently are integers from 1 to about 6, from 1 to about 4, or from1 to about 3. In some other embodiments, the small molecule linker is aderivative of 1,3-diamino-2-hydroxypropane. Some such derivatives havethe formula HO—(CH₂)_(m)—C(O)NH—CH₂—CH(OH)—CH₂—NHC(O)—(CH₂)_(m)—OH,wherein m is an integer from 0 to about 10, from 0 to about 6, from 2 toabout 6, or from 2 to about 4.

Some non-nucleotidic linkers according to the invention permitattachment of more than two oligoribonucleotides, as depicted inTable 1. For example, the small molecule linker glycerol has threehydroxyl groups to which oligoribonucleotides may be covalentlyattached. Some immune modulatory oligoribonucleotides according to theinvention, therefore, comprise more than two oligoribonucleotides (e.g.,a Domain C and so on, the additional domains compriseoligoribonucleotides as defined above for Domains A, B, C, and D) linkedat their 3′ ends to a non-nucleotidic linker.

In a further embodiment of this aspect of the invention, a SIMRA maycontain three or more oligoribonucleotides linked at their 3′ or 5′ends, or through an internucleoside linkage or a functionalizednucleobase or sugar to two or more linkers, as depicted in Table 1. Theoligoribonucleotides of this aspect of the invention may have the sameor different sequences. The linkers of this aspect of the invention maybe the same or different.

The immune modulatory oligoribonucleotides of the invention mayconveniently be synthesized using an automated synthesizer andphosphoramidite approach. In some embodiments, the immune modulatoryoligoribonucleotides are synthesized by a linear synthesis approach.

An alternative mode of synthesis is “parallel synthesis”, in whichsynthesis proceeds outward from a central linker moiety (see FIG. 1). Asolid support attached linker can be used for parallel synthesis, as isdescribed in U.S. Pat. No. 5,912,332. Alternatively, a universal solidsupport (such as phosphate attached controlled pore glass) support canbe used.

Parallel synthesis of immune modulatory oligoribonucleotides has severaladvantages over linear synthesis: (1) parallel synthesis permits theincorporation of identical monomeric units; (2) unlike in linearsynthesis, both (or all) the monomeric units are synthesized at the sametime, thereby the number of synthetic steps and the time required forthe synthesis is the same as that of a monomeric unit; and (3) thereduction in synthetic steps improves purity and yield of the finalimmune modulatory oligoribonucleotide product.

At the end of the synthesis by either linear synthesis or parallelsynthesis protocols, the immune modulatory oligoribonucleotides mayconveniently be deprotected with concentrated ammonia solution or asrecommended by the phosphoramidite supplier, if a modified nucleoside isincorporated. The product immune modulatory oligoribonucleotide ispreferably purified by reversed phase HPLC, detritylated, desalted anddialyzed.

Table 3 shows RNA-based immune modulatory oligoribonucleotides accordingto the invention. Unless otherwise specified, all nucleosides areribonucleosides.

TABLE 3 Stabilized RNA-based Immune Modulatory Oligonucleotide (SIMRA)Sequences SIMRA# (SEQ ID NO.) Sequences and Modification   15′-YUUCUG₁CUUCUG₁-X-G₁UCUUCG₁UCUUY-5′   25′-UG₁CUG₁CCUUUG₁-X-G₁UUUCCG₁UCG₁U-5′   35′-UG₁CUG₁CCUUUG₁-Z-G₁UUUCCG₁UCG₁U-5′   45′-G₁UCCUUUG₁CUG₁-X-G₁UCG₁UUUCCUG₁-5′   55′-L₁UGCUGCUUGUG-X-GUGUUCGUCGUL₁-5′   65′-LUGCUGCCUUUG-m-GUUUCCGUCGUL-5′   75′-G₁UCCUUG₁CUUG₁-M-G₁UUCG₁UUCCUG₁-5′   85′-UUCUG₁CUUCUG₁-M-G₁UCUUCG₁UCUU-5′   95′-G₁UCCUUUG₁CUG₁-m-G₁UCG₁UUUCCUG₁-5′  105′-YUUGACGUUGAC-m-CAGUUGCAGUUY-5′  11 5′-YGUGCCUGAUGA-X-AGUAGUCCGUGY-5′ 12 5′-AUGCUGCGCUG-M-GUCGCGUCGUA-5′  135′-UGCUGCUUG₂UG-X-GUG₂UUCGUCGU-5′  14 5′-UG₂CUGCUUGUG-X-GUGUUCGUCG₂U-5′ 15 5′-UG₂CUG₂CUUG₂UG₂-X-G₂UG₂UUCG₂UCG₂U-5′  165′-UG₂CUG₂CUUG₂UG₂-m-G₂UG₂UUCG₂UCG₂U-5′  175′-UG₂CUG₂CUUG₂UG₂-M-G₂UG₂UUCG₂UCG₂U-5′  185′-UG₂CUG₂CCUUUG₂-M-G₂UUUCCG₂UCG₂U-5′  195′-UG₂CUG₂CCUUUG₂-m-G₂UUUCCG₂UCG₂U-5′  205′-UG₂CUG₂CCUUUG₂-X-G₂UUUCCG₂UCG₂U-5′  215′-UGC₁UGC₁UUGUG-X-GUGUUC₁GUC₁GU-5′  225′-UGC₁UGC₁UUGUG-m-GUGUUC₁GUC₁GU-5′  235′-UGC₁UGC₁UUGUG-M-GUGUUC₁GUC₁GU-5′  245′-UGC₁UGC₁UUC₁UG-X-GUC₁UUC₁GUC₁GU-5′  255′-UGC₁UGC₁C₁UUUG-M-GUUUC₁C₁GUC₁GU-5′  265′-UGC₁UGC₁C₁UUUG-m-GUUUC₁C₁GUC₁GU-5′  275′-UGC₁UGC₁C₁UUUG-X-GUUUC₁C₁GUC₁GU-5′  285′-UGCUGCU₁U₁CU₁G-X-GU₁CU₁U₁CGUCGU-5′  295′-YUGCUGCU₁U₁CU₁G-X-GU₁CU₁U₁CGUCGUY-5′  305′-UGUUGUGUGA₁C-X-CA₁GUGUGUUGU-5′  315′-UG₂CUG₂CUUG₂UG₂-m-G₂UG₂UUCG₂UCG₂U-5′  325′-UGC₁UGC₁UUGUG-m-GUGUUC₁GUC₁GU-5′  335′-UGC₁UGC₁C₁UUUG-X-GUUUC₁C₁GUC₁GU-5′  345′-GAUUGUGACGU-X-UGCAGUGUUAG-5′  35 5′-CUGAAGCUUGU-X-UGUUCGAAGUC-5′  365′-UG₂CUG₂CUUG₂UG₂-M-G₂UG₂UUCG₂UCG₂U-5′  375′-YUGCUGCUUGUG-X-GUGUUCGUCGUY-5′  385′-UG₁CUG₁CUUCUG₁-X-G₁UCUUCG₁UCG₁U-5′  395′-UG₃CUG₃CUUCUG₃-X-G₃UCUUCG₃UCG₃U-5′  405′-UG₃CUG₃CCUUUG₃-m-G₃UUUCCG₃UCG₃U-5′  415′-YUGACGAUGAGU-X-UGAGUAGCAGUY-5′  425′-UGCUGCU₁U₁CU₁G-X-GU₁CU₁U₁CGUCGU-5′  435′-UGC₁UGC₁C₁UUUG-m-GUUUC₁C₁GUC₁GU-5′  445′-UG₁CUG₁CUUCUG₁-6Eg-M-6Eg-G₁UCUUCG₁UCG₁U-5′  455′-YUGACGACGCUU-X-UUCGCAGCAGUY-5′  46 5′-YUGACGACUGCU-X-UCGUCAGCAGUY-5′ 47 5′-YUGACGACUUGC-X-CGUUCAGCAGUY-5′  485′-YUGCGCGAACUU-X₃-UUCAAGCGCGUY-5′  495′-AG₁UG₁UUUUCUG₁-X-G₁UCUUUUG₁UG₁A-5′  505′-UG₁CUG₁CUUUUG₁-X-G₁UUUUCG₁UCG₁U-5′  515′-UG₁UUG₁UUUG₁UG₁-X-G₁UG₁UUUG₁UUG₁U-5′  525′-UG₁AUG₁AAG₁CUU-X-UUCG₁AAG₁UAG₁U-5′  535′-YUGCUGCUUGAA-X-AAGUUCGUCGUY-5′  54 5′-YUUGACUGAUGA-X-AGUUGUCAGUUY-5′ 55 5′-UGCUGCUUUUG-X-GUUUUCGUCGU-5′  56 5′-UGUUGUUUGUG-X-GUGUUUGUUGU-5′ 57 5′-UGUUCGAACAC-X-CACAAGCUUGU-5′  58 5′-UUGACGUUGAC-X-CAGUUGCAGUU-5′ 59 5′-UUGACGUUGAC-Z-CAGUUGCAGUU-5′  60 5′-UUGACGUUGAC-M-CAGUUGCAGUU-5′ 61 5′-UUGACGUUGAC-m-CAGUUGCAGUU-5′  62 5′-GUGCCUGAUGA-X-AGUAGUCCGUG-5′ 63 5′-CCGAUGCCGAC-X-CAGCCGUAGCC-5′  64 5′-CCGAUGCAUCG-X-GCUACGUAGCC-5′ 65 5′-GUGCCUGAUGA-Z-AGUAGUCCGUG-5′  66 5′-GUGCCUGAUGA-M-AGUAGUCCGUG-5′ 67 5′-GUGCCUGAUGA-m-AGUAGUCCGUG-5′  68 5′-CCGAUGCCGAC-Z-CAGCCGUAGCC-5′ 69 5′-CCGAUGCCGAC-M-CAGCCGUAGCC-5′  70 5′-CCGAUGCCGAC-m-CAGCCGUAGCC-5′ 71 5′-CCGAUGCAUCG-Z-GCUACGUAGCC-5′  72 5′-CCGAUGCAUCG-M-GCUACGUAGCC-5′ 73 5′-CCGAUGCAUCG-m-GCUACGUAGCC-5′  74 5′-AGCACAACUGU-X-UGUCAACACGA-5′ 75 5′-AAAAAAAAAAA-X-AAAAAAAAAAA-5′  765′-YCACUGUUGAGA-X-AGAGUUGUCACY-5′  77 5′-YAACUGUUGACC-X-CCAGUUGUCAAY-5′ 78 5′-YCAACGACCUGU-X-UGUCCAGCAACY-5′  795′-CACUG₁UUG₁AG₁A-X-AG₁AG₁UUG₁UCAC-5′  805′-AACUG₁UUG₁ACC-X-CCAG₁UUG₁UCAA-5′  815′-CAACG₁ACCUG₁U-X-UG₁UCCAG₁CAAC-5′  82 5′-AUGCUGCGCUG-X-GUCGCGUCGUA-5′ 83 5′-AUGCUGCGCUG-Z-GUCGCGUCGUA-5′  84 5′-AUGCUGCGCUG-m-GUCGCGUCGUA-5′ 85 5′-AACUGUUGACC-X-CCAGUUGUCAA-5′  86 5′-CACUGUUGAGA-X-AGAGUUGUCAC-5′ 87 5′-GCACACUUGUU-X-UUGUUCACACG-5′  88 5′-UGUUGUGUGAC-X-CAGUGUGUUGU-5′ 89 5′-CCGAUGCAUCG-X-GCUACGUAGCC-5′  90 5′-AACGAACCGAC-X-CAGCCAAGCAA-5′ 91 5′-YCAACGACCUGU-X-UGUCCAGCAACY-5′  925′-LCAACGACCUGU-X-UGUCCAGCAACL-5′  93 5′-CGUUGUGAUGA-X-AGUAGUGUUGC-5′ 94 5′-ACGAUUGUGAC-X-CAGUGUUAGCA-5′  95 5′-ACUUUGACGAU-X-UAGCAGUUUCA-5′ 96 5′-CGAUGCGAUGA-X-AGUAGCGUAGC-5′  97 5′-ACGUCUGACGA-X-AGCAGUCUGCA-5′ 98 5′-AACUGCUGGAU-X-UAGGUCGUCAA-5′  99 5′-UUGGACUCCAG-X-GACCUCAGGUU-5′100 5′-UCGACUUCCAG-X-GACCUUCAGCU-5′ 101 5′-CCGACUUGGAC-X-CAGGUUCAGCC-5′102 5′-AAGACUGAACU-X-UCAAGUCAGAA-5′ 1035′-YUG₂CUG₂CCUUUG₂-X-G₂UUUCCG₂UCG₂UY-5′ 1045′-YUG₂CUG₂CCUUUG₂-M-G₂UUUCCG₂UCG₂UY-5′ 1055′-YUG₂CUG₂CCUUUG₂-m-G₂UUUCCG₂UCG₂UY-5′ 1065′-LUG₂CUG₂CCUUUG₂-X-G₂UUUCCG₂UCG₂UL-5′ 1075′-LUG₂CUG₂CCUUUG₂-M-G₂UUUCCG₂UCG₂UL-5′ 1085′-LUG₂CUG₂CCUUUG₂-m-G₂UUUCCG₂UCG₂UL-5′ 1095′-L₁UG₂CUG₂CCUUUG₂-X-G₂UUUCCG₂UCG₂UL₁-5′ 1105′-L₁UG₂CUG₂CCUUUG₂-M-G₂UUUCCG₂UCG₂UL₁-5′ 1115′-L₁UG₂CUG₂CCUUUG₂-m-G₂UUUCCG₂UCG₂UL₁-5′ 1125′-G₁UCCUUG₁CUUG₁-m-G₁UUCG₁UUCCUG₁-5′ 1135′-UUCUG₁CUUCUG₁-m-G₁UCUUCG₁UCUU-5′ 1145′-UG₁CUG₁CCUUUG₁-M-G₁UUUCCG₁UCG₁U-5′ 1155′-UG₁CCUUUG₁CUG₁-M-G₁UCG₁UUUCCG₁U-5′ 1165′-UG₁CUG₁CUUCUG₁-M-G₁UCUUCG₁UCG₁U-5′ 1175′-UG₁C₁UG₁C₁C₁UUUG₁-X-G₁UUUC₁C₁G₁UC1G1U-5′ 1185′-UG₁C₁UG₁C₁UUC₁UG₁-X-G₁UC₁UUC₁G₁UC1G1U-5′ 1195′-YUGCUGCUUCUG-6Eg-M-6Eg-GUCUUCGUCGUY-5′ 1205′-UGGCUUGACGC-X-CGCAGUUCGGU-5′ 121 5′-UGCUGCUUGAA-X-AAGUUCGUCGU-5′ 1225′-UGACGAUGAGU-X-UGAGUAGCAGU-5′ 123 5′-UGAUGAGGACU-X-UCAGGAGUAGU-5′ 1245′-UGUUGAGGAAC-X-CAAGGAGUUGU-5′ 125 5′-UGCGAGACUGC-X-CGUCAGAGCGU-5′ 1265′-GUUGAACGACU-X-UCAGCAAGUUG-5′ 127 5′-UGACUGAUGAC-X-CAGUAGUCAGU-5′ 1285′-UGUUGAACGAC-X-CAGCAAGUUGU-5′ 129 5′-UGAGCGUGAAC-X-CAAGUGCGAGU-5′ 1305′-UG₁G₁CUUG₁ACG₁C-X-CG₁CAG₁UUCG₁G₁U-5′ 1315′-UG₁ACG₁AUG₁AG₁U-X-UG₁AG₁UAG₁CAG₁U-5′ 1325′-UG₁UUG₁AG₁G₁AAC-X-CAAG₁G₁AG₁UUG₁U-5′ 1335′-UG₁UUG₁AACG₁AC-X-CAG₁CAAG₁UUG₁U-5′ 1345′-YUGCGAGACUGC-X-CGUCAGAGCGUY-5′ 135 5′-YUGAGCGUGAAC-X-CAAGUGCGAGUY-5′136 5′-YUUGAGCUGGAC-X-CAGGUCGAGUUY-5′ 1375′-YGUUGAGGAACU-X-UCAAGGAGUUGY-5′ 138 5′-YUGAUGAAGCUU-X-UUCGAAGUAGUY-5′139 5′-YUUGACGAUGAG-X-GAGUAGCAGUUY-5′ 1405′-YUUGUUGAACGA-X-AGCAAGUUGUUY-5′ 141 5′-YUUGAACGACUU-X-UUCAGCAAGUUY-5′142 5′-YUGAUGGAACGA-X-AGCAAGGUAGUY-5′ 1435′-UG₁CUG₁CCUUUG₁-M-G₁UUUCCG₁UCG₁U-5′ 1445′-UG₁CUG₁CCUUUG₁-Y-M-Y-G₁UUUCCG₁UCG₁U-5′ 1455′-UG₁CUG₁CCUUUG₁-3Eg-M-3Eg-G₁UUUCCG₁UCG₁U-5′ 1465′-UG₁CUG₁CCUUUG₁-4Eg-M-4Eg-G₁UUUCCG₁UCG₁U-5′ 1475′-UG₁CUG₁CCUUUG₁-6Eg-M-6Eg-G₁UUUCCG₁UCG₁U-5′ 1485′-YUGCUGCUUGUG-Y-M-Y-GUGUUCGUCGUY-5′ 1495′-YUGCUGCUUGUG-3Eg-M-3Eg-GUGUUCGUCGUY-5′ 1505′-YUGCUGCUUGUG-4Eg-M-4Eg-GUGUUCGUCGUY-5′ 1515′-CAACGAACCCU-X-UCCCAAGCAAC-5′ 152 5′-UGCUGCUGCUG-X-GUCGUCGUCGU-5′ 1535′-UGAAGCUUGAA-X-AAGUUCGAAGU-5′ 154 5′-UGAACGUGAAC-X-CAAGUGCAAGU-5′ 1555′-YUGC₄UGC₄UUGUG-X-GUGUUC₄GUC₄GUY-5′ 1565′-UG₁C₄UG₁C₄UUC₄UG₁-X-G₁UC₄UUC₄G₁UC₄G₁U-5′ 1575′-YUG₁ACG₁AUG₁AG₁U-X-UG₁AG₁UAG₁CAG₁UY-5′ 1585′-G₁UCCUUG₁CUUG₁-X₁-G₁UUCG₁UUCCUG₁-5′ 1595′-UUCUG₁CUUCUG₁-X₁-G₁UCUUCG₁UCUU-5′ 1605′-UG₁CCUUUG₁CUG₁-X₂-G₁UCG₁UUUCCG₁U-5′ 1615′-UG₁CUG₁CUUCUG₁-X₂-G₁UCUUCG₁UCG₁U-5′ 1625′-PUUCUG₁CUUCUG₁-m-G₁UCUUCG₁UCUUP-5′ 1635′-PUG₁CCUUUG₁CUG₁-M-G₁UCG₁UUUCCG₁UP-5′ 1645′-UG₁CUG₁CUUC₁UG₁-X₁-G₁UC₁UUCG₁UCG₁U-5′ 1655′-UG₁CUG₁CUUC₁UG₁-X₃-G₁UC₁UUCG₁UCG₁U-5′ 1665′-C₂UGAAGC₂UUGU-X-UGUUC₂GAAGUC₂-5′ 1675′-CU₂GAAGCU₂U₂GU₂-X-U₂GU₂U₂CGAAGU₂C-5′ 1685′-YAACUG₂UUG₂ACC-X-CCAG₂UUG₂UCAAY-5′ 1695′-L₁UG₂G₂CUUG₂ACG₂C-X-CG₂CAG₂UUCG₂G₂UL₁-5′ 1705′-YUGACGCUGACU-X-UGACGCUGACUY-5′ 171 5′-YUGACUGCGACU-X-UCAGCGUCAGUY-5′172 5′-YUGCGAACGCUU-X-UUCGCAAGCGUY-5′ 1735′-YUGCGACUGACU-X₃-UCAGUCAGCGUY-5′ 1745′-YUGCGCUGAACU-X₃-UCAAGUCGCGUY-5′ 1755′-YUGCUGACGACU-X₃-UCAGCAGUCGUY-5′ 1765′-YUGCUUGAACGC-X₃-CGCAAGUUCGUY-5′ 1775′-YUUGCUGAACGC-X₃-CGCAAGUCGUUY-5′ 1785′-UG₁CUG₁CCUUUG₁-Y-X-Y-G₁UUCCG₁UCG₁U-5′ 1795′-YUGUUGUGUGAC-X-CAGUGUGUUGUY-5′ 180 5′-YUGCUGCCUUUG-X-GUUUCCGUCGUY-5′181 5′-YUGCUGCUGCUG-X-GUCGUCGUCGUY-5′ 1825′-YUGUUGUGUGAC-Z-CAGUGUGUUGUY-5′ 183 5′-EUUGAACGACUU-X-UUCAGCAAGUUE-5′184 5′-EUGUUGUGUGAC-X-CAGUGUGUUGUE-5′ 1855′-EUGCUGCCUUUG-X-GUUUCCGUCGUE-5′ 186 5′-EUGCUGCUGCUG-X-GUCGUCGUCGUE-5′187 5′-U₁GCUGCUUGUG-X-GUGUUCGUCGU₁-5′ 1885′-U₁GCU₁GCUUGUG-X-GUGUUCGU₁CGU₁-5′ 1895′-U₁GCU₁GCU₁U₁GU₁G-X-GU₁GU₁U₁CGU₁CGU₁-5′ G₁ = 7-deaza-rG; G₂ = ara-G;G₃ = 7-deaza-ara-G; C₁ = ara-C; C₂ = 2′-F-C; C₄ = 5-methyl-C;A₁ = ara-A; U₁ = ara-U; U₂= 2′-F-U; M = cis, cis-cyclohexanetriollinker; m = = cis, trans-cyclohexanetr1ol; Z = 1,3,5-pentane triollinker; X = glycerol linker; X₁ = 1,2,4 butane triol linker;X₂ = cyanuric acid; X₃ = isobutanetriol linker; Y = 1,3-propanediol; L= 1,5-pentanediol; L₁= 1′,2′-dideoxyribose; 6Eg = hexaethylene glycollinker; 3Eg = triethylene glycol linker; 4Eg = tetraethylene glycollinker; P = phosphorothioate; E = ethylane d1ol.

In a second aspect, the invention provides pharmaceutical formulationscomprising a SIMRA compound according to the invention and apharmaceutically acceptable carrier.

In a third aspect, the invention provides methods for generating TLR7and/or TLR8 mediated immune responses in a vertebrate, such methodscomprising administering to the vertebrate a SIMRA compound according tothe invention. In some embodiments, the vertebrate is a mammal. Inpreferred embodiments, SIMRA compound is administered to a vertebrate inneed of immune modulation.

In a fourth aspect, the invention provides methods for therapeuticallytreating a patient having a disease or disorder, such methods comprisingadministering to the patient a SIMRA compound according to theinvention. In various embodiments, the disease or disorder to be treatedis one in which an immune modulation may be desirable. For example, butnot limited to, cancer, an autoimmune disorder, infectious disease,airway inflammation, inflammatory disorders, allergy, asthma, or adisease caused by a pathogen. Pathogens include bacteria, parasites,fungi, viruses, viroids and prions.

In a fifth aspect, the invention provides methods for preventing adisease or disorder, such methods comprising administering to thepatient SIMRA compound according to the invention. In variousembodiments, the disease or disorder to be prevented is one in which animmune modulation may be desirable. For example, but not limited to,cancer, an autoimmune disorder, airway inflammation, inflammatorydisorders, infectious disease, allergy, asthma, or a disease caused by apathogen. Pathogens include bacteria, parasites, fungi, viruses,viroids, and prions.

In a sixth aspect the invention provides a method of preventing ortreating a disorder, such methods comprises isolating cells capable ofproducing cytokines or chemokines including, but not limited to, immunecells, B cells, T-regulatory cells, B-cells, PBMCs, pDCs and lymphoidcells; culturing such cells under standard cell culture conditions,treating such cells ex vivo with a SIMRA such that the isolated cellsproduce or secrete increased levels of cytokines or chemokines, andadministering or re-administering the treated cells to a patient in needof cytokine or chemokine therapy for the prevention or treatment ofdisease. This aspect of the invention would be in accordance withstandard adoptive cellular immunotherapy techniques to produce activatedimmune cells.

In some embodiments of this aspect of the invention, the cells capableof producing cytokines or chemokines may be isolated from subjects withor without a disease or disorder. Such isolation may includeidentification and selection and could be performed using standard cellisolation procedures, including those set forth in the specific examplesbelow. Such isolated cells are cultured according to standard cellculturing procedures and using standard cell culture conditions, whichmay include the culturing procedures and conditions set forth in thespecific examples below. In a further aspect of this embodiment of theinvention, the isolated cells are cultured in the presence of at leastone SIMRA, in an amount and for a time period sufficient to induce,increase or enhance the production and/or secretion of cytokines and/orchemokines as compared to the isolated cells cultured in the absence ofsuch one or more SIMRA. Such time may be from minutes, to hours, todays. Such isolated, SIMRA-treated cells may find use followingre-administration to the donor or administration to a secondhistologically compatible patient, wherein such donor or second patientare in need of induced, increased or enhanced production and/orsecretion of cytokines and/or chemokines. For example, re-administrationto a donor or administration to a second patient having cancer, anautoimmune disorder, airway inflammation, inflammatory disorders,infectious disease, allergy, asthma, or a disease caused by a pathogen.Such re-administration or administration may be accomplished usingvarious modes, including catheter or injection administration or anyother effective route. This aspect of the invention may also find use inpatients who may have a limited or incomplete ability to mount an immuneresponse or are immune compromised (e.g. patient infected with HIV andbone marrow transplant patients). This aspect of the invention may alsofind use in combination with SIMRA administration to the patientadministered or re-administered the isolated, SIMRA-treated cells.

In any of the methods according to the invention, the SIMRA compound canvariously act by producing direct immune modulatory effects alone or incombination with any other agent useful for treating or preventing thedisease or condition that does not diminish the immune modulatory effectof the SIMRA compound. In any of the methods according to the invention,the agent(s) useful for treating or preventing the disease or conditionincludes, but is not limited to, vaccines, antigens, antibodies,preferably monoclonal antibodies, cytotoxic agents, allergens,antibiotics, siRNA, microRNA, antisense oligonucleotides, TLR agonist(e.g. agonists of TLR9 and/or agonists of TLR7 and/or agonists of TLR8),chemotherapeutic agents (both traditional chemotherapy and moderntargeted therapies), targeted therapeutic agents, activated cells,peptides, proteins, gene therapy vectors, peptide vaccines, proteinvaccines, DNA vaccines, adjuvants, and co-stimulatory molecules (e.g.cytokines, chemokines, protein ligands, trans-activating factors,peptides or peptides comprising modified amino acids), or combinationsthereof. For example, in the treatment of cancer, it is contemplatedthat the SIMRA compound may be administered in combination with one ormore chemotherapeutic compound, targeted therapeutic agent and/ormonoclonal antibody. Alternatively, the agent can include DNA vectorsencoding for antigen or allergen. Alternatively, the SIMRA compounds canbe administered in combination with other adjuvants to enhance thespecificity or magnitude of the immune response to the SIMRA compound.

In any of the methods according to the invention, administration ofSIMRA compound, alone or in combination with any other agent, can be byany suitable route, including, without limitation, parenteral, mucosaldelivery, oral, sublingual, transdermal, topical, inhalation,intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal,by gene gun, dermal patch or in eye drop or mouthwash form.Administration of the therapeutic compositions of SIMRA compound can becarried out using known procedures using a pharmaceutically effectiveamount and for periods of time effective to reduce symptoms or surrogatemarkers of the disease. For example, a pharmaceutically effective amountof a SIMRA compound for treating a disease and/or disorder could be thatamount necessary to alleviate or reduce the symptoms, or delay orameliorate a tumor, cancer, or bacterial, viral or fungal infection. Apharmaceutically effective amount for use as a vaccine adjuvant could bethat amount useful for boosting a subject's immune response to a vaccineor antigen. In the context of administering a composition that modulatesan immune response to a co-administered antigen, a pharmaceuticallyeffective amount of a SIMRA compound and antigen is an amount sufficientto achieve the desired modulation as compared to the immune responseobtained when the antigen is administered alone. The effective amountfor any particular application can vary depending on such factors as thedisease or condition being treated, the particular oligonucleotide beingadministered, the size of the subject, or the severity of the disease orcondition. One of ordinary skill in the art can empirically determinethe pharmaceutically effective amount of a particular oligonucleotidewithout necessitating undue experimentation.

When administered systemically, the therapeutic composition ispreferably administered at a sufficient dosage to attain a blood levelof SIMRA compound from about 0.0001 micromolar to about 10 micromolar.For localized administration, much lower concentrations than this may beeffective, and much higher concentrations may be tolerated. Preferably,a total dosage of SIMRA compound ranges from about 0.001 mg per patientper day to about 200 mg per kg body weight per day. It may be desirableto administer simultaneously, or sequentially a therapeuticallyeffective amount of one or more of the therapeutic compositions of theinvention to an individual as a single treatment episode.

The SIMRA compound may optionally be linked to one or more allergensand/or antigens (self or foreign), an immunogenic protein or peptide,such as keyhole limpet hemocyanin (KLH), cholera toxin B subunit, or anyother immunogenic carrier protein. SIMRA can also be used in combinationwith other compounds (e.g. adjuvants) including, without limitation, TLRagonists (e.g. TLR2 agonists and TLR9 agonists), Freund's incompleteadjuvant, KLH, monophosphoryl lipid A (MPL), alum, and saponins,including QS-21 and imiquimod, or combinations thereof.

The methods according to this aspect of the invention are useful formodel studies of the immune system. The methods are also useful for theprophylactic or therapeutic treatment of human or animal disease. Forexample, the methods are useful for pediatric and veterinary vaccineapplications.

The examples below are intended to further illustrate certain exemplarembodiments of the invention, and are not intended to limit the scope ofthe invention.

EXAMPLES Example 1 Immune Modulatory Oligoribonucleotide Synthesis

The immune modulatory oligoribonucleotides were chemically synthesizedusing phosphoramidite chemistry on automated DNA/RNA synthesizer.N-acetyl protected (Except U) 2′-O-TBDMS RNA monomers, A, G, C and U,were purchased from Sigma-Aldrich. 7-deaza-G, inosine was purchased fromChemGenes Corporation. 0.25M 5-ethylthio-1H-tetrazole, PAC-anhydride CapA and Cap B were purchased from Glen Research. 3% trichloroacetic acid(TCA) in dichloromethane (DCM) and 5%3H-1,2-Benzodithiole-3-one-1,1-dioxide (Beaucage reagent) were made inhouse.

Immune modulatory oligoribonucleotides were synthesized at 1-2 μM scaleusing a standard RNA synthesis protocol.

Cleavage and Base Deprotection

Immune modulatory oligoribonucleotides were cleaved from solid supportand the protecting groups of exo-cyclic-amines were removed inmethylamine and ammonium hydroxide solution. The resulting solution wasdried completely in a SpeedVac.

IE HPLC Purification

Immune modulatory oligoribonucleotides were purified by ion exchangeHPLC. Using Dionex DNAPac 100 column. Crude immune modulatoryoligoribonucleotide solution was injected into HPLC. Above gradient isperformed and the fractions were collected. All fractions containingmore than 90% desired product were mixed, and then the solution wasconcentrated to almost dry by RotoVap. RNAse-free water was added tomake final volume of 10 ml.

C-18 Reversed Phase Desalting

tC-18 Sep-Pak cartridge purchased from Waters was washed by passing with10 ml of acetonitrile followed by 10 ml of 0.5 M sodium acetate throughthe cartridge. Then 10 ml of immune modulatory oligoribonucleotidesolution was loaded on to the cartridge. Then 15 ml of water was used towash out the salt. The immune modulatory oligoribonucleotide was finallyeluted using 1 ml of 50% acetonitrile in water. The solution was placedin SpeedVac for 30 minutes. The remaining solution was filtered througha 0.2 micron filter and then was lyophilized. The solid was thenre-dissolved in RNAse free water to make the desired concentration. Thefinal solution was stored below 0° C. Oligoribonucleotides were analyzedfor purity by Capillary Electrophoresis, Ion Exchange HPLC and PAGEanalysis, and for molecular mass by MALDI-ToF mass spectrometry.

Example 2 Protocols for Assays with HEK293 Cells Expressing TLRs

HEK293 or HEK293XL/human TLR7 or HEK293 or HEK293XL/human TLR8 cells(Invivogen, San Diego, Calif.) were cultured in 48-well plates in 250μl/well DMEM supplemented with 10% heat-inactivated FBS in a 5% CO₂incubator.

Reporter Gene Transformation

HEK293 or HEK293XL cells stably expressing human TLR7 or TLR8(Invivogen, San Diego, Calif.) were cultured in 48-well plates in 250μl/well DMEM supplemented with 10% heat-inactivated FBS in a 5% CO₂incubator. At 80% confluence, cultures were transiently transfected with400 ng/ml of SEAP (secreted form of human embryonic alkalinephosphatase) reporter plasmid (pNifty2-Seap) (Invivogen) in the presenceof 4 μl/ml of lipofectamine (Invitrogen, Carlsbad, Calif.) in culturemedium. Plasmid DNA and lipofectamine were diluted separately inserum-free medium and incubated at room temperature for 5 minutes. Afterincubation, the diluted DNA and lipofectamine were mixed and themixtures were incubated at room temperature for 20 minutes. Aliquots of25 μl of the DNA/lipofectamine mixture containing 100 ng of plasmid DNAand 1 μl of lipofectamine were added to each well of the cell cultureplate, and the cultures were continued for 4 hours.

IMO-Treatment

After transfection, medium was replaced with fresh culture medium. TheHEK293 or HEK29XL cells expressing human TLR7 or TLR8 were stimulatedwith 0, 20, 50, 100, 150, 200, or 300 μg/ml of agonists of TLR7 or TLR8,SIMRAs, and the cultures were continued for 18 hours-20 hours. At theend of SIMRA treatment, 30 μl of culture supernatant was taken from eachtreatment and used for SEAP assay following manufacturer's protocol(Invivogen).

Seap (Secreted Form of Human Embryonic Alkaline Phosphatase) Assay

Briefly, culture supernatants were incubated with p-nitrophynylphosphate substrate and the yellow color generated was measured by aplate reader at 405 nm. The data are shown as fold increase in NF-κBactivity over PBS control. (Putta M R et al, Nucleic Acids Res., 2006,34:3231-8).

Example 3 Human Cell Culture Protocols Human PBMC Isolation

Peripheral blood mononuclear cells (PBMCs) from freshly drawn, healthyvolunteer blood (CBR Laboratories, Boston, Mass.) were isolated byFicoll density gradient centrifugation method (Histopaque-1077, Sigma).

Human pDC Isolation

Peripheral blood mononuclear cells (PBMCs) from freshly drawn healthyvolunteer blood (CBR Laboratories, Boston, Mass.) were isolated byFicoll density gradient centrifugation method (Histopaque-1077, Sigma).pDCs were isolated from PBMCs by positive selection using the BDCA4 cellisolation kits (Miltenyi Biotec) according to the manufacturer'sinstructions.

Human mDC Isolation

Peripheral blood mononuclear cells (PBMCs) from freshly drawn healthyvolunteer blood (CBR Laboratories, Boston, Mass.) were isolated byFicoll density gradient centrifugation method (Histopaque-1077, Sigma).Myeloid dendritic cells (mDCs) were isolated from PBMCs by positiveselection using the BDCA4 cell isolation kits (Miltenyi Biotec)according to the manufacturer's instructions.

Multiplex Cytokine Assays

Human PBMCs were plated in 48-well plates using 5×10⁶ cells/ml. pDCswere plated in 96-well dishes using 1×10⁶ cells/ml. The SIMRAs dissolvedin DPBS (pH 7.4; Mediatech) were added to a final concentration of 20,50, 100, 200 or 300 μg/ml or as indicated in the figures to the cellcultures. The cells were then incubated at 37° C. for 24 hr and thesupernatants were collected for luminex multiplex or ELISA assays. Theexperiments were performed in triplicate wells. The levels of IFN-α,IL-6, or TNF-α were measured by sandwich ELISA. The required reagents,including cytokine antibodies and standards, were purchased fromPharMingen.

Luminex multiplex assays were performed using Biosource human multiplexcytokine assay kits on Luminex 100/200 instrument and the data wereanalyzed using StarStation software supplied by Applied CytometrySystems (Sacramento, Calif.).

Example 4 In Vivo Cytokine Secretion in Mouse Model Treated with TLR9Agonist Compounds

C57BL/6 mice and BALB/c mice, 5-6 weeks old, were obtained from TaconicFarms, Germantown, N.Y. and maintained in accordance with IderaPharmaceutical's IACUC approved animal protocols. Mice (n=3) wereinjected subcutaneously (s.c) with individual stabilized immunemodulatory RNA-based oligonucleotides from Table 3 at 25 mg/kg (singledose). Serum was collected by retro-orbital bleeding 2 hr after immunemodulatory oligonucleotide administration and cytokine and chemokinelevels were determined by sandwich ELISA or Luminex multiplex assays.The results are shown in FIGS. 8A, 8B, 9A and 9B and demonstrate that invivo administration of SIMRA oligonucleotides according to the inventiongenerates unique cytokine and chemokine profiles. All reagents,including cytokine and chemokine antibodies and standards were purchasedfrom PharMingen. (San Diego, Calif.).

Example 5 Serum Stability Assay

Approximately 0.5 OD of exemplar SIMRA compounds from Table 3 wasindividually incubated in 1% human serum in PBS for 30 minute at 37° C.Following 30 minutes of incubation in 1% human serum, the SIMRA compoundwas analyzed on anion-exchange HPLC to determine the percentage offull-length SIMRA compound that remained as compared to the amount ofSIMRA compound present before serum treatment. The results are shown inFIGS. 10A-10H and demonstrate that chemical modifications according tothe invention made to RNA-based compounds can enhance their stability.

EQUIVALENTS

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

1. A SIMRA compound selected from the group consisting of SIMRA#1 thoughSIMRA#189.
 2. A composition comprising the SIMRA compound of claim 1 anda physiologically acceptable carrier.
 3. A method for generating animmune response in a vertebrate, the method comprising administering tothe vertebrate a SIMRA compound according to claim
 1. 4. A method fortherapeutically treating a vertebrate having a disease or disorder wheremodulating an immune response would be beneficial such method comprisingadministering to the vertebrate a SIMRA compound according to claim 1 ina pharmaceutically effective amount.
 5. The method of claim 4 where inthe disease or disorder is cancer, an autoimmune disorder, airwayinflammation, inflammatory disorder, infectious disease, skin disorder,allergy, asthma or a disease caused by a pathogen.
 6. The methodaccording to claim 4, further comprising administering one or morechemotherapeutic compounds.
 7. The method according to claim 4, furthercomprising administering a targeted therapeutic agent.
 8. The methodaccording to claim 4, further comprising administering an antibody. 9.The method according to claim 4, further comprising administering a DNAvaccine.
 10. The method according to claim 4, further comprisingadministering a protein vaccine.
 11. The method according to claim 4,further comprising administering a peptide vaccine.
 12. The methodaccording to claim 4, further comprising administering an antigen. 13.The method according to claim 4, further comprising an adjuvant.
 14. Amethod for prophylactically treating a vertebrate having a disease ordisorder where modulating an immune response would be beneficial suchmethod comprising administering to the vertebrate a SIMRA compoundaccording to claim 1 in a pharmaceutically effective amount.
 15. Themethod of claim 14 wherein the disease or disorder is cancer, anautoimmune disorder, airway inflammation, inflammatory disorders,infectious disease, skin disorders, allergy, asthma or a disease causedby a pathogen in a vertebrate.
 16. The method according to claim 15,further comprising administering one or more chemotherapeutic compounds.17. The method according to claim 15, further comprising administering atargeted therapeutic agent.
 18. The method according to claim 15,further comprising administering an antibody.
 19. The method accordingto claim 15, further comprising administering a DNA vaccine.
 20. Themethod according to claim 15, further comprising administering a proteinvaccine.
 21. The method according to claim 15, further comprisingadministering a peptide vaccine.
 22. The method according to claim 15,further comprising administering an antigen.
 23. The method according toclaim 15, further comprising administering an adjuvant.